罗布麻基因文库的构建

CONSTRUCTION OF GENETIC LIBRARY FOR DOGBANE

本文从罗布麻（Ａｐｏｃｙｎｕｍｖｅｎｅｔｕｍ）幼苗中提取了大分子量ＤＮＡ，经Ｓａｕ３Ａ部分酶解，从琼脂糖凝胶中回收１２－２２ｋｂ的＂目的＂ＤＮＡ片段，用ＢａｍＨＩ／ＥｃｏＲＩ双酶切入ＥＭＢＬ３ＤＮＡ制备克隆载体。＂目的＂ＤＮＡ片段与载体连接，体外包装，所得重组子数为３．７４×１０６ｐｆｕ，达到了建库要求的理论值。以番茄１，５－二磷酸核酮糖羧化酶／加氧酶（Ｒｕｂｉｓｃｏ）小亚基基因（ｒｂｃＳ）的ｃＤＮＡ为探针，用噬菌斑原位杂交法，斑点杂交法，从基因文库中选出４个含罗布麻ｒｂｃＳ基因的阳性克隆。

Vector DNA was prepared by double digestion of λEMBL3 DNA withBamHI / EcoRI. High molecular weight dogbane (Apocynum Venetum) was isolated frometiolated shoots and partially digested with San 3A. The dogbane insert DNA fragementswith the size of 12-22 kb were recovered from agarose gel and joined with theEMBL3 vectors. The recombinant molecules were packaged into viable phage particles invitro and the yield of recombinant phages was 3.74 × 106 pfu. This figure met with the theoretical value required for dogbane genomic library. The library was screened by using theprob of tomato rbc S-2 cDNA. Four positive clones were obtained.